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received: 18 February 2016 accepted: 21 July 2016 Published: 17 August 2016

Recombinant adenovirus of human p66Shc inhibits MCF-7 cell proliferation Xiaoshan Yang1,2,*, Rong Xu1,2,*, Yajun  Lin2, Yongzhan Zhen3, Jie Wei2, Gang Hu2 & Hongfan Sun1 The aim of this work was to construct a human recombinant p66Shc adenovirus and to investigate the inhibition of recombinant p66Shc adenovirus on MCF-7 cells. The recombinant adenovirus expression vector was constructed using the Adeno-X Adenoviral System 3. Inhibition of MCF-7 cell proliferation was determined by MTT. Intracellular ROS was measured by DCFH-DA fluorescent probes, and 8-OHdG was detected by ELISA. Cell apoptosis and the cell cycle were assayed by flow cytometry. Western blot were used to observe protein expression. p66Shc expression was upregulated in 4 cell lines after infection. The inhibitory effect of p66Shc recombinant adenovirus on MCF-7 cells was accompanied by enhanced ROS and 8-OHdG. However, no significant differences were observed in the cell apoptosis rate. The ratio of the cell cycle G2/M phase showed a significant increase. Follow-up experiments demonstrated that the expressions of p53, p-p53, cyclin B1 and CDK1 were upregulated with the overexpression of p66Shc. The Adeno-X Adenoviral System 3 can be used to efficiently construct recombinant adenovirus containing p66Shc gene, and the Adeno-X can inhibit the proliferation of MCF-7 cells by inducing cell cycle arrest at the G2/M phase. These results suggested that p66Shc may be a key target for clinical cancer therapy. p66Shc, a 66 kDa proto-oncogene Src homologous-collagen homologue (Shc) adaptor protein, is an important protein that regulates the levels of reactive oxygen species (ROS) and lifespan in mammals1,2. Reactive oxygen species are widely accepted as one of the main factors of the aging process. p66Shc knockout mice have a lifespan approximately 30% longer and demonstrated an enhanced resistance to oxidative stress1 and age-related pathologies, such as atherosclerosis3,4, endothelial disorders5, obesity-induced insulin resistance6, AGE (advanced glycation end products)-dependent glomerulopathy related to diabetes mellitus7,8, and ethanol-induced liver disease9. Over the past decade, it was also reported that p66Shc can inhibit cell proliferation though blocking the MAPK or ERK signaling pathways10,11. Its effect on tumor cells has attracted the attention of researchers. Recently, numerous investigations have demonstrated that p66Shc can also inhibit and kill tumor cells12–14. The study of its function and its mechanism of action is particularly important. At present, researchers have clarified the protective effect of lower levels of p66Shc on the organism mostly using genetic mutation or knockout mutation techniques15–19. To increase the level of p66Shc expression, only plasmid transfection methods can be utilized to import the exogenous gene into cell lines. Due to the limited gene transfection efficiency, further research of the function of p66Shc, especially in primary cells and in vivo studies, is restricted. In this study, we constructed a human p66Shc recombinant adenovirus expression vector (AdenoX-p66Shc) using the Adeno-X Adenoviral System 3, which was easy to use and had a high efficiency for recombinant reactions. We observed a significantly higher expression of p66Shc in AdenoX-p66Shc infected cells, including primary cells, indicating that this tool could potentially be used to research the function of p66Shc in vitro and in vivo in the future.

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Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Peking Union Medical College & Chinese Academy of Medical Sciences, Tianjin 300192, China. 2The key Laboratory of Geriatrics, Beijing Hospital &Beijing Institute of Geriatrics, Ministry of Health, Beijing, 100730, China. 3Department of Histology and Embryology, College of Basic Medical, Hebei United University, Tangshan, 063000, China. *​These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Y.L. (email: [email protected]) or H.S. (email: [email protected]) Scientific Reports | 6:31534 | DOI: 10.1038/srep31534

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Figure 1.  Construction and expression of human recombinant p66Shc adenovirus. (A) Schematic presentation of p66Shc homologous recombination with pAdenoX-CMV. CH1: collagen-homology region; PTB: phosphotyrosine-binding domain; SH2: Src-homology2 domain; PCMV IE: cytomegalovirus immediate early promoter; SV40 polyA: simian virus 40 polyA signals; ITR: inverted terminal repeat; AMP: ampicillin. (B) HEK293A, HUVECs, HeLa and MCF-7 cells were infected with Ad-p66Shc or negative control (NC) for 48 h. The expression of p66Shc protein was detected by Western blot.

Results

Construction and expression of human recombinant p66Shc adenovirus.  The human p66Shc gene was amplified with 15 bp extensions that are homologous to the ends of the linearized adenoviral vector (Fig. 1A). To further validate the efficiency of the recombinant p66Shc adenovirus, HEK293A, HUVECs, HeLa and MCF-7 cells were infected by AdenoX-p66Shc (Ad-p66Shc) or a negative control for 48 h. Western blot analysis revealed that the expression of p66Shc in all of the cells infected by AdenoX-p66Shc was dramatically increased compared with the negative control (Fig. 1B). These results indicated that we had successfully constructed the recombinant adenovirus containing the human p66Shc gene, and the recombinant adenovirus was capable of efficiently infecting different types of cells.

p66Shc inhibited the proliferation of MCF-7 cells.  To determine the effect of p66Shc on cell viability, MCF-7 cells were infected with Ad-p66Shc or a negative control. As shown in Fig. 2A, upregulation of p66Shc by Ad-p66Shc decreased cell viability by 23% at 80 MOI and by 40% at 160 MOI. The cells were cultured for different time (12, 24, 36, 48, 60 h) in the presence of 100 MOI Ad-p66Shc. MCF-7 cells demonstrated decreased cell proliferation over time (Fig. 2B). These results demonstrated that p66Shc could inhibit the proliferation of MCF-7 cells. Induction of oxidative stress and associated DNA damage in MCF-7 cells.  Intracellular ROS were stained with DCFH-DA, and the fluorescence in each group was assessed by flow cytometry as described in the Materials and Methods. p66Shc could increase ROS levels in MCF-7 cells in a dose-dependent manner (Fig. 3A), and the DNA oxidative damage product 8-OHdG was significantly increased after Ad-p66Shc infection (Fig. 3B). Upregulation of p66Shc induced the expression and phosphorylation of p53.  We were also interested in the change of p53 expression and its phosphorylation, which was dependent on our target protein p66Shc, according to our data (Fig. 4A). To determine the mechanism, the levels of proteins related to mitochondrial apoptosis, including Bax and Bcl-2, were examined in MCF-7 cells and showed no significant change (Fig. 4A). The result also shows that the phosphorylation of p66Shc (p-p66Shc) on the S36 residue increased significantly, but the ratio of p-p66Shc to p66Shc was not changed significantly. A flow cytometry assay also verified that the number of apoptotic cells was similar between the cells infected with Ad-p66Shc and the negative control (Fig. 4B). Together, these data indicated that overexpression of p66Shc could increase the level of p53, but mitochondrial apoptosis could not explain the inhibitory effect induced by p66Shc. Scientific Reports | 6:31534 | DOI: 10.1038/srep31534

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Figure 2.  p66Shc inhibited MCF-7 cell viability. (A) MCF-7 cells were infected with various concentrations of Ad-p66Shc or negative control (20, 40, 80, 160, or 320 MOI) for 48 h. (B) MCF-7 cells were infected with 100 MOI Ad-p66Shc for different amounts of time (12, 24, 36, 48, or 60 h), and cell viability was detected using the MTT method. The data represent the means ±​SEM, n=​6 independent experiments. *p 

Recombinant adenovirus of human p66Shc inhibits MCF-7 cell proliferation.

The aim of this work was to construct a human recombinant p66Shc adenovirus and to investigate the inhibition of recombinant p66Shc adenovirus on MCF-...
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